KR102308854B1 - The pharmaceutical composition for prevention and treatment of coronavirus disease-19 containing carbocyclic nucleosides derivatives - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19). Specifically, the present invention relates to a pharmaceutical composition which contains a carbocyclic nucleoside derivative represented by chemical formula A-1 or chemical formula A-2 or a pharmaceutically acceptable salt thereof.

Description

A pharmaceutical composition for preventing and treating coronavirus infection-19 comprising a carbocyclic nucleoside derivative

The present invention relates to a pharmaceutical composition for the prevention and treatment of coronavirus infection-19 containing a carbocyclic nucleoside derivative, and specifically, has excellent infection inhibition ability and low cytotoxicity against SARS-CoV-2, so that the coronavirus It relates to a pharmaceutical composition comprising a carbocyclic nucleoside derivative that can be used to prevent or treat Infectious Disease-19.

Viruses are one of the leading causes of numerous incurable diseases that threaten human health, and enormous capital is being invested worldwide to prevent or treat them. Recently, coronavirus disease 19 (COVID-19), a respiratory infectious disease caused by a new type of severe acute respiratory syndrome coronavirus (SARS-CoV-2) that first occurred in Wuhan, China It has spread all over the world, causing a pandemic. Therefore, there is an urgent need to develop a therapeutic agent for viruses capable of inhibiting their proliferation, but currently there is no therapeutic or preventive agent capable of effectively and safely inhibiting these viruses.

On the other hand, antiviral agents can inhibit viruses through various mechanisms, among which the following two mechanisms are known to be used to effectively inhibit viruses.

The first is to inhibit S-adenosylhomocysteine (hereinafter, SAH) hydrolase. SAH hydrolase is a ^{tetramer-type enzyme that uses NAD +} as a coenzyme, and serves to reversibly hydrolyze SAH into adenosine (Ado) and homocysteine (Hcy), and It is a very important enzyme for the methylation of body substances such as histamine and norepinephrine as well as lipids and nucleic acids.

Inhibition of this SAH hydrolase causes the accumulation of SAH, and the excess SAH sequentially suppresses S-adenosylmethionine (AdoMet)-dependent transmethylase and capping of viral mRNA so that the protein required for viral replication is released. By preventing it from being made properly, it results in an antiviral effect.

Since most animal DNA viruses as well as RNA viruses require methylation enzymes (viral mRNA guanosine N7-methytransferases, O-2′-methytransferases) for mRNA capping, SAH hydrolase is considered an essential element in the development of broad-spectrum antiviral agents. do. In other words, it is considered that the development of RNA virus therapeutics and the development of SAH hydrolase inhibitors have a high correlation.

Second, it inhibits viral RNA polymerase. RNA viruses are replicated by inserting the substrate Nucleoside-5'-triphosphate (NTP) into the RNA chain by RNA polymerase. Therefore, a substance that inhibits RNA polymerase can also act as an antiviral agent, and it is converted into triphosphate in the body to selectively inhibit viral RNA polymerase or is directly inserted into the viral RNA chain to cause chain termination. ), it is considered that effective antiviral agents can be developed.

Tetrahedron: Asymmetry, 2002, 13, 1189-1193 J. Med. Chem. 2001, 44, 3985-3993

Accordingly, the problem to be solved by the present invention is to discover carbocyclic nucleoside derivatives having excellent infection inhibition ability and low cytotoxicity against SARS-CoV-2, a kind of coronavirus, which causes serious risks worldwide. To provide a pharmaceutical composition for preventing or treating coronavirus infection-19 (COVID-19).

The problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

A pharmaceutical composition for preventing and/or treating coronavirus infection-19 (COVID-19) according to an embodiment of the present invention for solving the above problems is a carbocyclic compound represented by the following Chemical Formula A-1 or Chemical Formula A-2 and a nucleoside (carbocyclic nucleoside) derivative or a pharmaceutically acceptable salt thereof.

<Formula A-1>

<Formula A-2>

In Formula A-1 or Formula A-2, R ₁ and R ₂ are each independently hydrogen or _{fluorine, but at least one of R 1} and R ₂ is fluorine, and B is represented by Formula B-1 below.

<Formula B-1>

In Formula B-1, X is a substituted or unsubstituted amino group, Y is hydrogen, a thiophenyl group, a furanyl group or a pyrrolyl group, and Z is hydrogen or a substituted or unsubstituted alkynyl group

The substituted amino group may be an alkyl amino group or an aryl amino group, and the substituted alkynyl group may be an alkyl alkynyl group or an aryl alkynyl group.

The alkylamino group is -NHMe, -NHEt, -NMe ₂ , -NMeEt, -NEt ₂ , benzylamino or halogenated benzylamino, and the arylamino group is phenylamino or halogenated phenylamino ( halogenized phenylamino), the alkyl alkynyl group may be benzylethynyl or benzylpropynyl, and the arylalkynyl group may be phenylethynyl or phenylpropynyl.

The halogenated phenylamino group or halogenated benzylamino group may be, for example, fluorinated (fluoro), chlorinated (chloro), brominated (bromo) or iodinated (iodo) phenylamino or benzylamino, but is not limited thereto.

The alkynyl group may be, for example, ethynyl, propynyl, or butynyl, but is not limited thereto.

Specifically, Formula A-1 may be Formula 1-1 or Formula 1-2, and Formula A-2 may be Formula 2 below.

<Formula 1-1>

<Formula 1-2>

<Formula 2>

The pharmaceutical composition for preventing and/or treating COVID-19 according to an embodiment of the present invention may include the above-described carbocyclic nucleoside derivative as an active ingredient against SARS-CoV-2. That is, embodiments of the present invention provide an antiviral agent or anti-SARS-CoV-2 agent for SARS-CoV-2 including the above-described carbocyclic nucleoside derivative. ) can be

Antiviral agent refers to a pharmaceutical composition that can be used not only for inhibiting virus activity, replication, etc., but also for preventing and/or treating all diseases caused by viruses.

The carbocyclic nucleoside derivative or salt may have excellent infection inhibition ability and low cytotoxicity against SARS-CoV-2. Specifically, the carbocyclic nucleoside derivative or salt may have an IC ₅₀ value for SARS-CoV-2 of 7.5 μM or less, a CC ₅₀ value of 50 or more, and/or an SI value of 6.5 or more. More specifically, the carbocyclic nucleoside derivative or salt may have an IC ₅₀ value of 4 μM or less and/or an SI value of 16 or more for SARS-CoV-2.

Alternatively, the carbocyclic nucleoside derivative or salt may have an inhibition of infection for SARS-CoV-2 of 70% or more at a concentration of 10 μM. At the same time, the viable cell ratio (cell ratio) may be greater than 70%. The infection inhibition rate may be a relative ratio derived compared to uninfected cells (infectivity 0%) and infected cells (infectivity 100%) that were not treated with the carbocyclic nucleoside derivative or salt.

The carbocyclic nucleoside derivative represented by Formula A-1 or Formula A-2 may be provided in the form of a pharmaceutically acceptable salt. As the salt, acid addition salts formed with various pharmaceutically acceptable organic or inorganic acids are useful. Suitable organic acids include, for example, carboxylic acid, phosphonic acid, sulfonic acid, acetic acid, propionic acid, octanoic acid, decanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, malic acid, tartaric acid, citric acid, glutamic acid, aspartic acid, Maleic acid, benzoic acid, salicylic acid, phthalic acid, phenylacetic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, etc. can be used. For example, halogen acids such as hydrochloric acid and sulfuric acid or phosphoric acid can be used. can

However, the present invention is not limited thereto, and the carbocyclic nucleoside derivative of the present invention may be provided in the form of all salts, hydrates and solvates that can be prepared by conventional methods.

The pharmaceutical composition for preventing and/or treating COVID-19 may be administered systemically or locally, and may include fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. that can be generally used for oral or parenteral administration. It may be formulated using an excipient (or diluent). Hereinafter, excipients and formulation methods are specifically illustrated, but not limited to these examples.

Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid dosage forms include at least one excipient, for example, in one or more of the compounds of Formula A-1 or Formula A-2. For example, it can be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid formulations for oral administration include, for example, suspensions, internal solutions, emulsions, syrups, and the like, and the liquid formulation includes various excipients, such as wetting agents, in addition to commonly used simple diluents such as water and liquid paraffin, sweetening agents, flavoring agents, preservatives, and the like.

Formulations for parenteral administration may include injections, emulsions, inhalants, suppositories, and the like. Injections may include sterile aqueous solvents, non-aqueous solvents and suspensions, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, esters such as ethyl oleate, etc., and suppositories include witepsol, Macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be included. In addition, for topical application, the antiviral agent of the present invention may be formulated as an ointment or cream.

The preferred dosage of the pharmaceutical composition varies depending on a number of factors such as the patient's condition and weight, the degree of disease, the drug form, the route and duration of administration, and the like, but may be appropriately selected by those skilled in the art. In addition, the route of administration may vary depending on the patient's condition and its severity.

The details of other embodiments are included in the detailed description.

Carbocyclic nucleoside derivatives according to embodiments of the present invention are safe and effective for preventing and treating coronavirus infection-19 (COVID-19) because they have excellent infection inhibition and low cytotoxicity against SARS-CoV-2. can be utilized.

Effects according to the embodiments of the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

1 is a flowchart illustrating a SARS-CoV-2 infection analysis process through HTS automation according to an experimental example of the present invention.
2 is a map (Plate map) in which each well of the 384-tissue culture plate according to the experimental example of the present invention is divided by treatment material and method.
3 is a diagram illustrating a principle of analyzing an image using IM software according to an experimental example of the present invention.
4 is an image of heatmap analysis of each plate treated according to an experimental example of the present invention.
5 to 7 are graphs showing the inhibition of infection and cytotoxicity of each well treated with compounds according to the experimental example of the present invention.
8 is a confocal microscope image of wells treated with 10 μM of the three types of compounds finally screened according to an experimental example of the present invention and control wells.
9 is a graph analyzing the performance and Z'-factor of control wells according to an experimental example of the present invention.
10 is a graph showing a dose response curve (DRC) derived through analysis according to an experimental example of the present invention.

Advantages and features of the present invention, and methods of achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the embodiments allow the disclosure of the present invention to be complete, and those of ordinary skill in the art to which the present invention pertains. It is provided to fully inform the person of the scope of the invention, and the present invention is only defined by the scope of the claims.

The terminology used herein is for the purpose of describing the embodiments and is not intended to limit the present invention. In this specification, 'and/or' includes each and every combination of one or more of the mentioned items. The singular also includes the plural, unless the phrase specifically states otherwise. As used herein, 'comprises' and/or 'comprising' does not exclude the presence or addition of one or more other elements in addition to the stated elements. Numerical ranges indicated using '-' or 'to' indicate a numerical range including the values listed before and after as the lower and upper limits, respectively, unless otherwise stated. 'About' or 'approximately' means a value or numerical range within 20% of the value or numerical range recited thereafter.

In addition, in describing the components of the embodiment of the present invention, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only for distinguishing the components from other components, and the essence, order, or order of the components are not limited by the terms.

Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used with the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not to be interpreted ideally or excessively unless clearly defined in particular.

And in describing the embodiment of the present invention, if it is determined that a detailed description of a related known configuration or function interferes with the understanding of the embodiment of the present invention, the detailed description thereof will be omitted.

As used herein, 'prevention' refers to suppressing the occurrence of symptoms or diseases in an individual who has no symptoms or diseases yet, but may be afflicted with such symptoms or diseases.

As used herein, 'treatment' refers to (a) inhibition of the development (exacerbation) of a symptom or disease, (b) alleviation or amelioration of the symptom or disease, or (c) elimination of the symptom or disease in an individual.

As used herein, the term 'subject' refers to animals, particularly mammals, including humans, having symptoms or diseases that can be 'prevented' or 'treated' by administering the composition of the present invention.

Hereinafter, embodiments of the present invention will be described in detail through Preparation Examples and Experimental Examples, but it is apparent that the effect of the present invention is not limited by the following Experimental Examples.

Preparation Example: Preparation of the carbocyclic nucleoside derivative of the present invention

Preparation Example 1: Formula 1-1

<substance 1>

Method for the preparation of substance 1: References [1] Tetrahedron: Asymmetry, 2002, 13, 1189-1193 and [2] J. Med. Chem. It was synthesized with reference to 2001, 44, 3985-3993.

<Substance 2a-2c>

Method for preparing materials 2a and 2b: The previously synthesized material 1 (6 g, 24.8 mmol) was dissolved in anhydrous THF, chlorotriethylsilane (TESCl) (16.7 mL, 99.2 mmol) was added dropwise and cooled to -78 °C. . To the cooled mixture, a 1.0 M lithium bis(trimethylsilyl)amide/THF solution (LiHMDS 1.0 M solution in THF) (50 mL, 50 mmol) was slowly added dropwise, and the reaction was stirred well under the same conditions for 30 minutes, followed by silyl enol ether. make you angry After TLC confirmed that the reaction was complete, the reaction mixture was quenched with a saturated aqueous ammonium chloride (NH ₄ Cl) solution, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, _{dried over magnesium sulfate (MgSO 4} ), and the residual solid was removed by filtration under reduced pressure and then concentrated under reduced pressure. The concentrated residue was dissolved in anhydrous DMF, and then cooled to 0 °C. To the cooled mixture was added 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo [2.2.2] octane bis(tetrafluoroborate) (trade name Selectfluor ^® ) (10.15 g, 28.65 mmol) 1.5 When an equivalent is added dropwise and stirred for 15 hours under the same conditions, materials 2a and 2b, which are fluorinated compounds, are produced in a ratio of 3:1, respectively (confirmed by NMR as a ratio). When it is confirmed by TLC that the reaction is complete, the reaction mixture is quenched with a saturated aqueous ammonium chloride (NH ₄ Cl) solution, the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, _{dried over magnesium sulfate (MgSO 4} ), filtered under reduced pressure to remove the residual solid, and then concentrated under reduced pressure. The concentrated residue is separated and purified through silica gel chromatography, etc. to obtain 2a (6.591 g, 58%) and 2b (3.078 g, 27%), respectively.

2a: ¹ H NMR (400 MHz, CDCl ₃ ) 5.29 (dd, J = 8.2, 49.5 Hz, 1 H), 4.70 (t, J = 5.7 Hz, 1 H), 4.20 (dd, J = 2.4, 6.1 Hz) , 1 H), 3.61 (dd, J = 1.6, 8.6 Hz, 1 H) 3.38-3.41 (m, 1 H), 2.75 (d, J = 8.2 Hz, 1 H), 1.41 (s, 3 H), 1.30 (s, 3 H), 1.06 (s, 9 H)

2b: ¹ H NMR (600 MHz, CDCl ₃ ) 5.21-5.36 (ddd, J = 1.3, 4.5, 50.8 Hz, 1 H,), 4.55 (d, J = 5.9 Hz, 1 H), 4.50 (d, J) = 5.9 Hz, 1 H), 3.63 (d, J = 2.2 Hz, 2 H), 2.52-2.58 (m, 1 H), 1.41 (s, 3 H), 1.33 (s, 3 H), 1.13 (s) , 9 H)

Method for the preparation of material 2c: 2c (2.95 g, 84%) can be synthesized by performing the same synthesis of material 2a (3.29 g, 12.6 mmol) synthesized above from material 1 to materials 2a and 2b.

¹ H NMR (500 MHz, CDCl ₃ ) 4.72 (t, J = 6.1 Hz, 1 H), 4.35-4.38 (m, 1 H), 3.68 (d, J = 8.6 Hz, 1 H), 3.68 (d, J = 8.6 Hz, 1 H) 3.46 (d, J = 8.5 Hz, 1 H), 2.67 (d, J = 17.4 Hz, 1 H), 1.42 (s, 3 H), 1.33 (s, 3 H), 1.05 (s, 9 H). (mixed with a peak presumed to be a substance equilibrated in the form of a diol)

<Substance 3a-3c>

Method for preparing material 3a: After dissolving material 2a (3.33 g, 12.8 mmol) in methanol, it is sufficiently cooled to 0° C. or less. To the cooled solution is _{slowly added sodium borohydride (NaBH 4} ) (1.45 g, 38.4 mmol). Thereafter, the reaction is stirred under the same conditions, the completion of the reaction is confirmed by TLC, the reaction mixture is terminated with a saturated aqueous ammonium chloride (NH ₄ Cl) solution, the aqueous layer is extracted with ethyl acetate, and the organic layer is separated. The separated organic layer was washed thoroughly with water and brine, _{dried over magnesium sulfate (MgSO 4} ), filtered under reduced pressure to remove the residual solid, and then concentrated under reduced pressure. An intermediate (2.54 g, 76%) obtained by separation and purification of the alcohol compound produced by silica gel chromatography, etc. is dissolved in anhydrous pyridine, and then cooled to 0 °C. To the cooled mixture, trifluoromethanesulfonic anhydride (3.26 ml, 19.36 mmol) was added dropwise, and stirred under the same conditions for 30 minutes to oxidize trifluoromethylsulfonic acid. After confirming the completion of the reaction by TLC, the reaction mixture was quenched with a saturated _{aqueous solution of ammonium chloride (NH 4} Cl), the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, _{dried over magnesium sulfate (MgSO 4} ), filtered under reduced pressure to remove the residual solid, and then concentrated under reduced pressure. The resulting trifluoromethylsulfonated compound and sodium azide (NaN ₃ ) (1.89 g, 29.04 mmol) were mixed in anhydrous DMF, and stirred under heating at 60° C. to azide. After stirring for about 4 hours, the reaction was confirmed by TLC, the reaction was terminated with water, the aqueous layer was extracted with ethyl acetate, and the organic layer was separated. The separated organic layer was washed thoroughly with water and brine, _{dried over magnesium sulfate (MgSO 4} ), filtered under reduced pressure to remove the residual solid, and then concentrated under reduced pressure. The obtained azide compound is separated and purified through silica gel chromatography, etc., and the obtained compound (4.07 g, 42%) is dissolved in methanol. An appropriate amount of palladium/carbon is added to the solution, the reaction vessel is replaced with hydrogen, and the hydrogenation reaction proceeds to reduce the azide group to an amine group. After confirming the completion of the reaction by TLC, when the reaction is completed, the remaining solid is removed through reduced pressure filtration and concentrated under reduced pressure to obtain the desired material 3a. The obtained material 3a proceeds to the next process without separation and purification.

¹ H NMR (600 MHz, CDCl ₃ ) d 4.96 (dt, J =52.5, 3.3 Hz, 1 H), 4.31-4.36 (m, 2 H), 3.48-3.54 (m, 2 H), 3.27 (td, J = 4.1, 28.4 Hz, 1 H), 2.24-2.34 (m, 1 H), 1.80 (s, 2 H), 1.45 (s, 3 H), 1.26 (s, 3 H), 1.16 (s, 9) H)

Preparation method of material 3c: 3c (2.98 g, 60%) can be synthesized by proceeding with the same procedure for the synthesis of material 2a to material 3a as described above for material 2c (4.95 g, 17.79 mmol) synthesized above. However, in the reaction of alcoholizing ketones, lithium borohydride (LiBH _{4 ) is used instead of sodium borohydride (NaBH 4} ), and 10 equivalents of sodium azide is used in the azide reaction and the temperature is set to 100 °C. should be raised, and the stirring time should be increased to about 15 hours.

¹ H NMR (800 MHz, CDCl ₃ ) d 4.45-4.46 (m, 1 H), 4.25-4.27 (m, 1 H), 3.58 (dd, J = 4.5, 9.2 Hz, 1 H), 3.54 (dd, J = 5.1, 9.2 Hz, 1 H), 3.35-3.38 (m, 1 H), 2.56-2.59 (m, 1 H), 1.94 (bs, 3 H), 1.47 (s, 3 H), 1.28 (s) , 3 H), 1.18 (s, 9 H)

<Substance 4a-4c>

Preparation of Substance 4a: Substance 3a (982 mg, 3.757 mmol) synthesized above and 5-amino-4,6-dichloropyrimidine (1.85 g, 11.27 mmol), di diisopropylethylamine (DIPEA) (6.54 mL, 37.57 mmol) of n - and heated to 170 ℃ using that later, microwave, etc. mixed in butanol (n -butanol), and the mixture was stirred for 4 hours. After confirming that the reaction is complete by TLC, the reaction mixture is mixed with methanol and concentrated under reduced pressure, and the residue is separated and purified by silica gel chromatography, etc. to obtain an intermediate (964 mg, 66%). This intermediate was dissolved in diethoxymethyl acetate and stirred at 140° C. for about 3 hours using a microwave or the like. After confirming the completion of the reaction by TLC, the reaction mixture is mixed with methanol, concentrated under reduced pressure, and separated and purified by silica gel chromatography, etc. to obtain material 4a (643 mg, 65%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.74 (s, 1 H), 8.34 (d, J = 2.4 Hz, 1 H), 5.28-5.43 (dt, J = 52.8, 2.8 Hz, 1 H), 5.12 -5.23 (m, 2 H), 4.61 (t, J = 5.0 Hz, 1 H), 3.65-3.69 (m, 1 H), 3.61 (t, J = 9.2 Hz, 1 H), 2.56-2.71 (m) , 1 H), 1.56 (s, 3 H), 1.32 (s, 3 H), 1.17 (s, 9 H)

Method for the preparation of material 4c: 4c (223 mg, 50%) can be synthesized by using the above-synthesized material 3c (182 mg, 0.625 mmol) in the same manner as for the synthesis of material 4a from material 3a. However, in the first reaction step, the temperature should be raised to 200 °C and the stirring time should be increased to about 7 hours.

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.76 (s, 1 H), 8.76 (s, 1 H), 8.29 (d, J = 2.4 Hz, 1 H), 5.26-5.37 (m, 1 H), 5.11 (t, J = 6.9 Hz, 1 H), 4.59-4.64 (m, 1 H), 3.64-3.74 (m, 2 H), 2.81-2.96 (m, 1 H), 1.58 (s, 3 H) , 1.33 (s, 3 H), 1.20 (s, 9 H)

<Formula 1-1>

Preparation method of Formula 1-1: After dissolving the material 4a (220 mg, 0.54 mmol) synthesized above in tert-butanol ( tert- butanol), it is transferred to a high-temperature airtight container (stainless steel bomb reactor). After adding dropwise saturated ammonia/tert-butanol (tertiary butanol) to the mixture, the reaction vessel is sealed and stirred at 120° C. for about 15 hours. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with methanol and then concentrated under reduced pressure. After the concentrated residue is dissolved in THF, a solution of trifluoroacetic acid (TFA) and water in a ratio of 2:1 is added dropwise to the mixture. After the reaction mixture was stirred at 60 °C for about 15 hours, the completion of the reaction was confirmed by TLC and concentrated under reduced pressure. The concentrated residue is separated and purified by silica gel chromatography, etc. to obtain the final compound of Formula 1-1 (66 mg, 43%).

¹ H NMR (400 MHz, CD ₃ OD) δ 8.31 (d, J = 2.4 Hz, 1 H), 8.23 (s, 1 H), 5.36-5.39 (m, 1 H), 5.20-5.35 (td, J) = 2.8, 53.6 Hz, 1 H), 5.05-5.16 (m, 1 H), 4.66-4.69 (m, 1 H), 3.64-3.71 (m, 2 H), 2.51-2.66 (m, 1 H), 1.55 (s, 3 H), 1.35 (s, 3 H), 1.22 (s, 9 H)

Preparation Example 2: Formula 1-2

<Formula 1-2>

Compound 4c (223 mg, 0.534 mmol) synthesized above can be synthesized in the same manner as in the synthesis of material 4a to Formula 1-1, as described above, to synthesize the compound of Formula 1-2 (99 mg, 61%).

¹ H NMR (400 MHz, CD ₃ OD) δ 8.29 (s, 1 H), 8.21 (s, 1 H), 5.29-5.36 (m, 2 H), 4.66 (bs, 1 H), 3.75-3.79 ( m, 1 H), 3.66-3.70 (m, 1 H), 2.80-2.89 (m, 1 H), 1.57 (s, 3 H), 1.34 (s, 3 H), 1.22 (s, 9 H)

Preparation Example 3: Formula 2

<Substance 5>

Preparation method of substance 5: 2,2,6,6-tetramethylpiperidine (TMP, 59.2 g, 419 mmol) was dissolved in anhydrous THF (170 mL) under nitrogen gas and n-BuLi (285 mL, 2.5 M in) at -78 ° C. hexane, 457 mmol) was slowly added and stirred at 0 °C for 2 hours. The reaction mixture was lowered to -78 °C again, and the starting material 6-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (20 g, 84 mmol) dissolved in anhydrous THF (110 mL) was added. Add slowly and stir for 3 hours. To this reaction mixture, iodine (102 g, 402 mmol) dissolved in THF (200 mL) was slowly added at -78 °C, stirred at the same temperature for 3 hours, and then slowly raised to 0 °C over 5 hours. After confirming the completion of the reaction, add 10% sodium thiosulfate solution (550 mL) and saturated NH4Cl aqueous solution (250 mL) to terminate the reaction. The reaction mixture is extracted with EtOAc (500 mL), dried over anhydrous MgSO4, and concentrated under reduced pressure. The remaining residue is purified by flash column chromatography under the condition of CH2Cl2/MeOH = 100/1 to obtain the desired compound 5 (66%) in the form of an ivory solid.

¹ H NMR (600 MHz, CDCl ₃ ) δ 5.66 (dd, J = 2.4, 10.8 Hz, 1H), 4.20 (m, 1 H), 3.74 (td, J = 2.4, 12 Hz, 1H), 3.04 (m , 1 H), 2.17 (m, 1 H), 1.62-1.91 (m, 4 H).

<Substance 6>

Method for preparing material 6: Starting material 5 (500 mg, 1.01 mmol) was well dissolved in anhydrous DMF (10 mL) and toluene (10 mL), and then Bis(dibenzylideneacetone)palladium(0) (57.5 mg, 0.10 mmol), Add copper(I) iodide (38.4 mg, 0.20 mmol), diisopropylamine (306.60 mg, 3.03 mmol), and phenylacetylene (1.1 equiv.), and stir thoroughly. The reaction mixture was stirred for 12 hours at room temperature under nitrogen gas. Upon completion of the reaction, the reaction mixture is extracted with EtOAc (20 mL), dried over anhydrous MgSO4, and concentrated. The residue is purified by column chromatography under the condition of hexanes/EtOAc = 4/1 to obtain the target compound 6 (75%).

¹ H NMR (600 MHz, CDCl ₃ ) δ 7.66-7.64 (m, 2H), 7.52-7.42 (m, 3H), 5.88 (dd, J = 11.3, 2.1 Hz, 1H), 4.23 (d, J = 11.5) Hz, 1H), 3.80-3.74 (m, 1H), 2.92 (qd, J = 12.1, 4.0 Hz, 1H), 2.17-2.14 (m, 1H), 1.96-1.55 (m, 4H).

<Substance 7>

Preparation method of material 7: Dissolve starting material 6 in anhydrous THF (0.13 M), add bis(triphenylphosphine)palladium(II) dichloride, 0.1 equivalents and 2-tributylstannylthiophene, 2 equivalents, and stir at room temperature under nitrogen gas substitution conditions. After heating and stirring the above mixture at 60 °C for 3 hours, the temperature was lowered to room temperature and evaporated. The remaining material is separated by column chromatography (silica gel, hexanes/EtOAc, 15/1) to obtain a final product 7.

¹ H NMR (600 MHz, DMSO) δ (ppm): 7.90 (dd, 1 H, J = 8.0, 6.8 Hz), 7.43 (m, 2 H), 7.46 (m, 3 H), 7.42 (dd, 1 H, J = 3.6, 0.8 Hz), 7.11 (dd, 1 H, J = 3.6, 1.6 Hz), 5.79 (dd, 1 H, J = 11.2, 2.4 Hz), 4.09 (d, 1 H, J = 11.2) Hz), 3.70 (m, 1 H), 3.50 (m, 1H), 2.69 (t, 2 H, J = 6.8 Hz), 2.03 (m, 2 H), 1.74 (m, 1 H).

<Substance 8>

Preparation method of material 8: Anhydrous ethanol (0.13 M) was added to the starting material 7 and stirred, and 0.2 equivalents of pyridinium p-toluenesulfonate was added at room temperature under nitrogen gas substitution. After stirring at the same temperature for 4 hours, triethylamine (1 mL) was added to terminate the reaction, and then evaporated. The reaction product was separated by column chromatography to obtain a final product 8 (60%).

¹ H NMR (600 MHz, CDCl ₃ ) δ 8.06 (d, J = 3.2 Hz, 1H), 7.51-7.35 (m, 6H), 7.13 (t, J = 4.3 Hz, 1H).

<Substance 9>

Preparation method of material 9: After sufficiently dissolving the starting material 8 (0.5 mmol) in EDC (5 mL), N,O-bis(trimethylsilyl)acetamide (BSA) (1.44 equivalents) is slowly added dropwise at room temperature. After the reaction mixture was stirred at 40 °C for 1 hour, sugar 3 (1.2 eq) was dissolved in EDC (2 mL) at room temperature and slowly added dropwise. After the reaction mixture was cooled to 0 °C, TMSOTf (0.68 equiv) was added, the temperature was raised to 80 °C, and the mixture was stirred for 3 hours. When the reaction is completed, the reaction mixture is treated _{with a saturated aqueous NaHCO 3} solution and extracted _{with CH 2} Cl _{2 .} The residue is concentrated and purified by column chromatography to obtain the target compound 9 (58%).

¹ H NMR (600 MHz, CDCl ₃ ) δ 8.04 (d, J = 3.2 Hz, 1H), 7.66 (d, J = 6.9 Hz, 2H), 7.51-7.42 (m, 4H), 7.15 (t, J = 4.3 Hz, 1H), 6.41 (s, 2H), 5.95 (s, 1H), 4.93 (dd, J = 10.5, 4.1 Hz, 1H), 4.27 (d, J = 10.5 Hz, 1H), 2.22 (s, 3H), 2.08 (s, 3H).

<Formula 2>

Preparation method of Formula 2: Anhydrous ethanol (0.3 M) was added to the starting material 9 and stirred, then trimethylamine (4 equivalents) and methylamine hydrochloride (1.3 equivalents) were added and stirred at room temperature under nitrogen gas filling. After stirring at the same temperature for 24 hours and evaporating, the resulting mixture is separated by column chromatography to obtain the final product, Chemical Formula 2 (Substance 10).

¹ H NMR (600 MHz, MeOD) δ 7.87 (dd, J = 1.0, 3.6 Hz, 1 H), 7.63-7.67 (m, 2 H), 7.41-7.50 (m, 4 H), 7.08 (dd, J) = 3.6, 5.0 Hz, 1 H), 6.22 (d, J = 6.0 Hz, 1 H), 5.46 (dd, J = 4.7, 5.8 Hz, 1 H), 4.72 (dd, J = 3.3, 9.5 Hz, 1 H), 4.05 (dd, J = 1.3, 9.5 Hz, 1 H), 3.17 (s, 3 H).

Experimental Example: Activity test for SARS-CoV-2

In order to verify the activity of the compounds of the present invention against SARS-CoV-2, the compounds were tested in a coronavirus evaluation model. To this end, using Image Mining (IM) software, confocal microscopy images of viral N protein and cell nuclei were analyzed, and dose response curves (DRCs) were generated for each compound. 1 is a flowchart showing the SARS-CoV-2 infection analysis process through HTS automation according to this experimental example, and the specific experimental method was as follows.

SARS-CoV-2 was provided from the Korea Centers for Disease Control and Prevention (KCDC), and Vero cells were obtained from ATCC.

^{1.2x10 4} Vero cells per well were seeded in a 384-tissue culture plate. After 24 hours, the cells were treated with 50 μM of the compounds prepared at 10 points by serial dilution twice in DMSO at the highest concentration. Figure 2 is a map (Plate map) in which each well of the 384-tissue culture plate is divided by treatment material and method. In FIG. 2, blue 'Compound' wells represent wells treated with 84 compounds including the compounds of the present invention. The above process was equally performed on two plates (Replicate 1 and 2) for reliability verification.

About 1 hour after compound treatment, cells were infected with 0.0125 MOI of SARS-CoV-2 in a BSL3 facility and incubated at 37°C for 24 hours. After fixing the cells with 4% paraformaldehyde (PFA), permeabilization was performed. Thereafter, cells were stained with anti-SARS-CoV-2 Nucleocapsid (N) primary antibody and 488-conjugated goat anti-rabbit IgG secondary antibody and Hoechst 33342. Fluorescence expression was imaged using a large-capacity image analysis device, Operetta (Perkin Elmer).

The acquired images were analyzed using an internal analysis program, Image Mining (IM) software. 3 is a diagram illustrating a principle of analyzing an image using IM software.

The total number of cells per well was calculated as the number of Hoechst-stained nuclei, and the number of infected cells was calculated as the number of cells expressing the nucleocapsid protein. The infectivity (infection ratio) was calculated as the number of cells expressing the nucleocapsid protein/total number of cells. Infectivity per well was defined as 0% of the average infectivity of wells containing uninfected cells (mock) in the same plate and 100% of the average infectivity of wells containing untreated infected cells (0.5% DMSO group) in the same plate. was normalized. The response curve and IC ₅₀ and CC ₅₀ values according to drug concentration were derived using the formula Y = Bottom + (Top Bottom)/(1 + (IC ₅₀ /X)Hillslope) using XLFit 4 (IDBS) software. All IC ₅₀ and CC ₅₀ values were measured in two replicates, and the reliability of the assay was verified with the values of Z'-factor and percent coefficient of variation (%CV).

4 is an image of heatmap analysis of each plate processed according to the above experimental example. 5 to 7 are graphs showing the inhibition of infection and cytotoxicity of each well treated with the compounds.

Through the image analysis result of FIG. 4 and the graphs of FIGS. 5 to 7, five compounds (Positives + Toxic-Positives) having an infection inhibition ability of 70% or more were screened, among which the viable cell ratio showing cytotoxicity was 70 % or more of three compounds (Positives) were screened. 8 is a confocal microscope image of wells treated with 10 μM of the final screened three compounds (FM4L-038, FM2L-001, FM2L-002) and control wells. FM2L-001, FM2L-002 and FM4L-038 represent compounds of Formulas 1-1, 1-2 and 2, respectively.

On the other hand, the graph showing the correlation between the two plates in FIGS. 5 and 6 supports that the experiment was performed reliably. 9 is a graph analyzing the performance and Z'-factor of the control wells, where the Z'-factor is obtained through the mean values and standard deviations of positive and negative controls, and is 0.5 or more. In the case of , it can be judged as an experiment with established reliability. Since the Z' factor of the above experiment was 0.82, it can be seen that the experiment was well verified.

10 is a graph showing a dose response curve (DRC) derived through the analysis, and Table 1 below shows IC ₅₀ , CC ₅₀ and SI values derived through DRC of each compound. As shown in FIG. 10 , the final screened compounds of the present invention are significantly higher than the existing compounds known to be effective against SARS-CoV-2, such as chloroquine, remdesivir and lopinavir. It shows a low IC ₅₀ value, showing excellent infection inhibition, and at the same time showing an equivalent level of CC ₅₀ value and thus a relatively high SI value, it can be used as a safer and more effective SARS-CoV-2 infectious disease (COVID-19) prevention and treatment. can

IC ₅₀ (μM) CC ₅₀ (μM) SI Formula 1-1 7.43 > 50 6.73 Formula 1-2 3.08 > 50 16.25 Formula 2 7.22 > 50 6.93 Chloroquine 9.71 > 150 15.44 Remdesivir 7.90 > 50 6.33 Lopinavir 20.77 > 50 2.41

In the above, the embodiment of the present invention has been mainly described, but this is only an example and does not limit the present invention. It will be appreciated that various modifications and applications not exemplified above are possible. For example, each component specifically shown in the embodiment of the present invention may be implemented by modification. And differences related to such modifications and applications should be construed as being included in the scope of the present invention defined in the appended claims.

Claims (8)

A carbocyclic nucleoside derivative represented by the following formula (2) or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for preventing or treating Coronavirus Infectious Disease-19 (COVID-19).
<Formula 2>

delete delete delete delete delete According to claim 1,
The carbocyclic nucleoside derivative or salt has an IC ₅₀ value of 7.5 μM or less _{for SARS-CoV-2, a CC 50} value of 50 or more, and an SI value of 6.5 or more.
A pharmaceutical composition for preventing or treating COVID-19. 8. The method of claim 7,
The carbocyclic nucleoside derivative or salt has an IC ₅₀ value of 4 μM or less and an SI value of 16 or more for SARS-CoV-2.
A pharmaceutical composition for preventing or treating COVID-19.

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